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Infection, Genetics and Evolution 11 (2011) 789–797 Contents lists available at ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid Review Bacillus taxonomy in the genomic era finds phenotypes to be essential though often misleading Heather Maughan a,1,*, Geraldine Van der Auwera b,c,1 a Department of Cell & Systems Biology, University of Toronto, 25 Willcocks St., Toronto, ON, Canada M5S 3B2 Department of Microbiology & Molecular Genetics, Harvard Medical School, MA, USA c Laboratory of Food and Environmental Microbiology, Université catholique de Louvain, Louvain-la-Neuve, Belgium b A R T I C L E I N F O A B S T R A C T Article history: Received 14 September 2010 Received in revised form 31 January 2011 Accepted 1 February 2011 Available online 18 February 2011 Bacillus is a diverse bacterial genus characterized by cells growing aerobically and forming dormant endospores. Although Bacillus species were some of the first bacteria ever characterized, their relationships to one another remain enigmatic. The recent deluge of environmental sequencing projects has further complicated our view of Bacillus taxonomy and diversity. In this review we discuss the current state of Bacillus taxonomy and focus on two examples that highlight the ecological diversity found within identical 16S rDNA-based clusters: the identification of ecologically distinct clusters of B. simplex in Evolution Canyons and the demarcation of species in the industrially and medically important B. cereus group. These examples highlight the difficulties of purely 16S rDNA-based taxonomy, emphasizing the need to interpret the massive amounts of molecular data from environmental sequencing projects in a bacterial ecology framework. Such interpretations are likely to reveal ecological diversity within Bacillus that extends beyond that previously imaginable, providing a true picture of Bacillus ecology and evolution. ! 2011 Elsevier B.V. All rights reserved. Keywords: Bacillus Taxonomy 16S rDNA Bacillus cereus sensu lato Phylogenetics Bacterial species Contents 1. 2. 3. 4. 5. Introduction . . . . . . . . . . . . . . . . . . . . . . . . Historical overview of Bacillus taxonomy . Modern Bacillus taxonomy . . . . . . . . . . . . Limitations of 16S taxonomy . . . . . . . . . . 4.1. B. simplex . . . . . . . . . . . . . . . . . . . . . 4.2. B. cereus group . . . . . . . . . . . . . . . . Reconciling molecules and ecology. . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction The genus Bacillus is comprised of low G+C Gram positive bacteria (Kingdom Bacteria; Phylum Firmicutes; Class Bacilli; Order Bacillales; Family Bacillaceae) and is most closely related to the genera Listeria, Streptococcus, and Staphylococcus (Ciccarelli et al., 2006; Wu et al., 2009). Bacillus species are ubiquitous in * Corresponding author. Tel.: +1 416 946 7121. E-mail address: [email protected] (H. Maughan). 1 These authors contributed equally to this work. 1567-1348/$ – see front matter ! 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2011.02.001 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 789 790 791 792 792 792 795 795 795 nature, having been isolated from environments as diverse as freshwater, saline water, soil, plants, animals, and air (Pignatelli et al., 2009). The phenotypic diversity encompassed by members of the Bacillus genus is spectacular: high temperatures, extreme salinity, acidic conditions and the immune systems of many animals pose little challenge to some members (Holt, 1986). Some species use unusual terminal electron acceptors like arsenic or selenium (Switzer Blum et al., 1998), have increased growth in the presence of activated charcoal (Fujita et al., 1996), and are capable of colonizing environments as peculiar as paintings (Taubel et al., 2003). The metabolic breadth of Bacillus has been harnessed by industry for the production of molecules such as riboflavin, 790 H. Maughan, G. Van der Auwera / Infection, Genetics and Evolution 11 (2011) 789–797 Streptavidin, b-lactamase, and a diversity of insect and nematode toxins (de Maagd et al., 2003; Zeigler and Perkins, 2009). At least two Bacillus species, B. cereus and B. anthracis, infect humans causing food-borne illness and anthrax, respectively. These examples illustrate the usefulness of some Bacillus species and the danger of others. However, as we discuss in this review, resolving Bacillus isolates into neatly defined species with welldefined boundaries is not straightforward. The classification of strains and species in an appropriate manner is vital for the responsible production of industrially important molecules and the effective control of insect and animal pathogens. Moreover, the exponential growth of genomic databases from environmental sequencing projects will require a firm ecological and evolutionary framework in which to classify Bacillus sequences and appropriately define new species. The need for efficient classification has been made apparent by recent research suggesting that Bacillus species boundaries defined by gene sequence data are not always in agreement with boundaries defined by ecology; we discuss this issue below in the context of B. cereus and its close relatives. Bacillus is not unique in this respect, as classifications of other genera have similar issues. Thus, the current challenge, in bacterial taxonomy in general and Bacillus taxonomy in particular, is to find a systematic way to reconcile molecular data with ecology for a valid and biologically meaningful classification (Achtman and Wagner, 2008; Cohan, 2002; Fox et al., 1992; Koeppel et al., 2008). We highlight these issues in Bacillus and discuss the implications for the levels of ecological diversity likely present, based on environmental sequencing projects. One lineage that suitably illustrates the disagreement between molecular and phenotypic/ecological methods of classification in Bacillus is the B. cereus group. This group, also called B. cereus sensu lato, contains six very closely related species according to current taxonomy: B. cereus, B. thuringiensis, B. anthracis, B. mycoides, B. pseudomycoides, and B. weihenstephanensis. Over the past century or so, these six species were described as individual species of the Bacillus genus using pathogenic host range, colony morphology and metabolic properties as distinguishing criteria, along with motility, resistance to penicillin and sensitivity to gamma phage. However, molecular methods have since shown that the species boundaries between members of this group are difficult to define, forcing us to rethink our current descriptions of these and other Bacillus species. 2. Historical overview of Bacillus taxonomy Bacillus species such as B. anthracis and B. subtilis were among the first species of bacteria ever described in a systematic manner in the late 19th century by premier German biologists Ferdinand Cohn and Robert Koch (Cohn, 1962; Koch, 1962). Cohn was first to discover sporulation in B. subtilis (Cohn, 1962) and Koch famously proved the germ theory of disease using B. anthracis (Koch, 1962), the causative agent of anthrax and early biological warfare agent. Cohn and Koch classified bacteria based on cell shape (sphericals, short rods, threads, and spirals). The use of such crude morphological criteria, imposed by the technological limitations of the time, led to an overly simplistic and often misleading classification that failed to reflect relationships of common ancestry between organisms. For example, Escherichia coli and Pseudomonas aeruginosa were classified as species of Bacillus by virtue of their rod-shaped cells (Daegelen et al., 2009; Hugh and Leifson, 1964). Furthermore, use of the ability to form spores as a defining criterion resulted in the inclusion in the Bacillus genus of a considerable diversity of bacteria that have since been reassigned into different genera (Ash et al., 1991, 1993; Shida et al., 1996). In the 20th century, the study of bacteria blossomed from simple microscopic observations to the detailed study of cell structure and physiology. In addition to providing the first glimpse of molecular biology, these studies enabled the characterization of any bacterial culture using a series of biochemical, physiological, and morphological tests, e.g., catalase enzyme production, carbon source utilization, lipid composition, and the presence and positioning of flagella. These characterizations led to a formalized classification in reference books such as Bergey’s manual (Holt, 1986), which microbiologists could use as a key to identify novel isolates of interest. Using such criteria Bacillus was defined as Gram-positive, aerobic, motile, spore-forming rods that produce catalase; additional tests were used to further characterize species within the Bacillus genus. Although biochemical testing was a very useful tool that yielded invaluable details about function and ecology, it suffered from two major drawbacks. In practice, despite being relatively easy to perform, the biochemical tests were difficult to standardize between laboratories and could only be performed on bacteria that would grow under ordinary laboratory conditions. This method of classification would therefore be inapplicable to fastidious or uncultured organisms, which are estimated to represent a huge proportion of the true bacterial diversity to be found in nature. But most importantly, the functional nature of such tests meant that they yielded little information concerning the genetic basis for the abilities being probed, and potentially misleading information in terms of evolutionary relatedness. Indeed, between any two isolates, shared abilities could be due to evolutionary convergence or, in the case of shared genetic material, horizontal gene transfer. Conversely, shared disabilities could be due to independent gene loss events or differential regulation. Therefore, phenotypic similarities cannot be taken with certainty to indicate close evolutionary relatedness. During the heyday of phenotype-based taxonomy, the DNA of many isolates was also compared using pairwise whole genome hybridization techniques. These techniques were based on comparing hybridization kinetics for DNA of a single strain vs. DNA of a pair of divergent strains; DNA of a single strain will hybridize quickly whereas divergent DNA will take longer to hybridize, the more divergent the longer, or in the case of unique regions of DNA, will not hybridize at all. Although hybridization techniques enabled the quantification of genetic distance between different isolates, they were only useful for very closely related species. The contribution of molecular genetics to bacterial taxonomy really flourished when American biologist Carl Woese showed that the gene sequence encoding the ubiquitous 16S rRNA gene of the ribosomal subunit could be used to reconstruct phylogeny on an unprecedented scale (Olsen and Woese, 1993; Woese et al., 1990), finally making it possible to classify bacterial species in an evolutionary framework. 16S rDNA sequencing seemed at the time to be the perfect solution since it is relatively inexpensive, fully reproducible between laboratories, does not require culturing of the bacteria and it is a universal molecule. The general outcome of Woese’s work was an evolution-based view of organismal taxonomy that divided prokaryotes into eubacteria and archaea and joined these with eukaryotes to result in the three domains of life (Woese et al., 1990). It also enabled a fairly detailed mapping of phylogeny within eubacterial genera, including a considerable amount of remapping and re-assigning species to different genera than what the original identifications made using classical criteria had dictated. 16S rDNA-based taxonomy seemed like a clear way forward, except in the case of closely related species groups such as Bacillus, where insufficient divergence in 16S rDNA prevented the resolution of strain and species relationships. Subsequent use of housekeeping genes that are essential and thus not lost from genomes, but that evolve more quickly than 16S rDNA, has proven to be useful for taxonomic classification (Palys et al., 2000, 1997). H. Maughan, G. Van der Auwera / Infection, Genetics and Evolution 11 (2011) 789–797 791 Fig. 1. Evolutionary relationships of 59 Bacillus species inferred using (A) Maximum Likelihood analysis of the 16S rDNA locus and (B) rearrangement of branches in A to minimize the number of evolutionary changes in the following 11 phenotypes: maximum growth temperature, minimum growth temperature, Voges–Proskauer test, ability to grow anaerobically, acid production from glucose, acid production from arabinose, acid production from mannitol, hydrolysis of starch, flagella present, spore shape, and sporangium swelling. Numbers on the molecular tree indicate support from 1000 bootstrap pseudoreplicates in FastTree (Price et al., 2009, 2010). Tree branch rearrangements were done in Mesquite (Maddison and Maddison, 2009) using the ‘‘Search for a Better Tree’’ function. Listeria monocytogenes was chosen as the outgroup because it is a well-supported sister group of Bacillus (Ciccarelli et al., 2006; Wu et al., 2009). Phenotypic information was obtained from the following sources: (Ajithkumar et al., 2002; Arfman et al., 1992; Boone et al., 1995; Boyer et al., 1973; Combet-Blanc et al., 1995; De Clerk et al., 2004; Fujita et al., 1996; Gordon et al., 1977; Heyrman et al., 2003; Holt, 1986; Kanso et al., 2002; Kuhnigk et al., 1995; Lechner et al., 1998; Li et al., 2002; Logan et al., 2000, 2002; Nagel and Andreesen, 1991; Nakamura, 1989, 1998; Nakamura et al., 1988, 1999; Nielsen et al., 1995; Palmisano et al., 2001; Pettersson et al., 2000, 1996; Pichinoty et al., 1983; Priest et al., 1987, 1988; Reva et al., 2002; Roberts et al., 1994, 1996; Spanka and Fritze, 1993; Switzer Blum et al., 1998; Venkateswaran et al., 2003; Yoon et al., 2001, 2003; Yumoto et al., 2003; Zhou et al., 2008). Although such approaches are useful for single isolates studied intensively in the laboratory, 16S rDNA remains the gold standard for environmental sequencing projects due to its ubiquity and ease of amplification from divergent species (e.g., Pignatelli et al., 2009). To illustrate how taxonomic relationships may differ between phenotypic and 16S rDNA-based inferences, we inferred the relationships of 59 Bacillus species using 16S rDNA sequences, and subsequently altered the topology to minimize changes in 11 phenotypes (see Fig. 1 legend for details). These phenotypes were chosen based on their early use to classify Bacillus in Bergey’s manual, and subsequent species descriptions were used for additional phenotype information (see Fig. 1 legend for references). As shown in Fig. 1, relationships between species are drastically different when phenotypic information is used to refine the tree topology. For example, the clade containing the wellstudied B. subtilis subsp. subtilis (blue font in Fig. 1) has high support in the molecular tree, but when phenotypic information is used to rearrange branches, most species in this clade are dispersed throughout the tree. However, many of the closest relatives of B. subtilis subsp. subtilis in the molecular tree (B. mojavensis, B. pumilus, B. vallismortis, and B. atrophaeus) maintain each other as close relatives in the phenotypic tree. The retention of closely related groups is also seen with the B. cereus group, where most of these species are close relatives in both trees. This indicates that when strains/species have low 16S rDNA divergence, many of their phenotypes are likely to be similar, but the converse is not true: strains/species with similar phenotypes are not necessarily closely related at the 16S rDNA level. This concept is not novel; although there are disadvantages to using only the 16S rDNA locus for phylogenetic reconstruction (Forney et al., 2004), the underlying idea that molecular data should be the basis of modern bacterial taxonomy has become largely uncontroversial. 3. Modern Bacillus taxonomy What have 16S rDNA data told us about Bacillus? Sequencing 16S rDNA from environmental samples has given us a renewed appreciation of the diversity of Bacillus. To evaluate this diversity we inferred a new Bacillus tree using 16S rDNA sequences available in the Ribosomal Database Project (RDP; Cole et al., 2009); these sequences correspond to both cultured, well-studied Bacillus species as well as 16S rDNA sequences obtained by direct sequencing of environmental samples. 7510 sequences were aligned and their relationships inferred with Maximum Likelihood in FastTree using 16S rDNA sequences from the closely related genus Listeria for an outgroup (Price et al., 2009, 2010). Sequence identity cutoffs were implemented in TreeChopper (http://microbiomeutil.sourceforge.net/). We found that using the conventional 792 H. Maughan, G. Van der Auwera / Infection, Genetics and Evolution 11 (2011) 789–797 previous phenotype-based classifications. In the next section we present two examples. 4. Limitations of 16S taxonomy 16S rDNA-based taxonomy suffers from the major drawback that ecology does not inform species designations and cutoff values are essentially arbitrary. We present two examples where consideration of ecologically important phenotypes leads to a significantly improved species grouping than obtained using only 16S rDNA sequencing. In the first example we discuss how B. simplex lineages with the same 16S rDNA sequence correspond to divergent ecotypes. In the second example, we discuss how some very closely related lineages of bacteria in the B. cereus group are designated as different species despite having the same 16S rDNA, for historical reasons and partly because their ecologies have repercussions on human affairs. Although 16S-rDNA based taxonomy has failed to identify ecologically important clusters in these species groups, molecular techniques focused on more rapidly evolving portions of the genome have identified such clusters, or ecotypes, in B. simplex. This has proven to be more difficult in the B. cereus group. Fig. 2. Bacillus phylogeny based on Maximum Likelihood analysis of 16S sequences listed as Bacillus (or Listeria for outgroup) in the Ribosomal Database Project as of July 2009. The inner most ring shows the branches indicating evolutionary relatedness and divergence. These branches are extended with dashes to the species names (black ring fanning out towards outside of circle). Colors indicate the following: blue: B. cereus group; yellow: B. subtilis; green: uncultured; aqua: Listeria outgroup. No color indicates a previously described Bacillus species outside the B. subtilis and B. cereus groups. Species names are as assigned in the RDP database. Numbers refer to the species groups discussed in the main text (1: B. subtilis group; 2: B. cereus group; 3: uncultured or previously described Bacillus species). cutoff of 97% identity (Stackebrandt and Goebel, 1994) predicts the existence of 116 species of Bacillus. Fig. 2 shows the evolutionary relationships of the 7510 16S rDNA sequences. Culture-associated sequences designated as B. subtilis (yellow labels and clade 1 in Fig. 2) or belonging to the B. cereus group (blue labels and clade 2 in Fig. 2) dominate the 16S rDNA phylogeny, which is easily explained by the extensive sampling and sequencing efforts that focus on these groups. The interesting observation here is that despite the disproportionately large amount of sampling, the 16S rDNA diversity within these groups is low, as evidenced by the shallow branch lengths that populate them. This marks a striking contrast to the generally much longer branch lengths extending to 16S rDNA sequences from uncultured environmental samples not belonging to B. subtilis or B. cereus (green labels and clade 3 in Fig. 2), and described species represented by only one or a few sequences (no label and clade 3 in Fig. 2). Crucially, these longer branch lengths underscore the tremendous diversity present outside of the two well-studied groups, B. subtilis and B. cereus, greatly extending previous descriptions of this diversity (Priest, 1993; Zeigler and Perkins, 2009). Furthermore, although most sequences from uncultured samples are clustered together with other previously described Bacillus species that are not members of the subtilis or cereus groups, over one hundred are actually interspersed within the B. subtilis and B. cereus groups. This strongly suggests that although these groups are thought to be very well-studied, there may be some fundamental aspects of their diversity that still defy our understanding. Although 16S rDNA sequencing has been exceptionally powerful for taxonomical classification in an evolutionary framework, it has also highlighted some major issues with 4.1. B. simplex Nevo and colleagues have spent years measuring ecological divergence between strains of B. simplex living in different habitats within three ‘‘Evolution Canyons’’ (EC) in Israel (Sikorski and Nevo, 2005, 2007). The north facing slope, valley bottom, and south facing slope within each EC differ in their soil characteristics and the UV and sunlight intensities they receive, but these environmental parameters are comparable between the corresponding areas of the ECs (Fig. 3). This consistently replicated pattern of divergent ecological conditions within a relatively small geographic space has provided an excellent system in which to address ecological divergence and sympatric speciation. Hundreds of strains were isolated from each of the three canyon environments and characterized at the genetic and phenotypic levels. Although all strains were identical at the 16S rRNA gene, distinct genetic profiles could be distinguished using Random Amplified Polymorphic DNA-PCR fingerprinting techniques and sequencing of housekeeping genes. Phenotypic profiles were then drawn by measuring UV survival, mutation rates, and growth at high temperature for each strain. Comparison of phenotypic and genetic profiles showed that strains from similar ecologies were more similar to each other than were strains from adjacent geographical areas. For example, strains living on the hotter south facing slopes of each of the three canyons were more similar to each other, regardless of canyon of origin, than to strains on the opposite slopes of their respective canyons of origin (Fig. 3). Simulations predicting ecologically distinct groups of strains were used with sequence data from housekeeping genes for a subset of strains (Koeppel et al., 2008). These simulations predicted ecologically distinct lineages that corresponded nicely with the phenotype data, indicating that lineages with different ecologies are diverging at the genetic level. Because ecologically distinct populations are not subject to each other’s selective sweeps, divergence can occur at the genetic level (Cohan, 2002; Koeppel et al., 2008). Taken together, these results show that even though all B. simplex strains share the same 16S rDNA sequence, they are adapted to very different ecologies. 4.2. B. cereus group B. cereus, B. anthracis, B. mycoides and B. thuringiensis were described very early on in the history of the Bacillus genus, on the H. Maughan, G. Van der Auwera / Infection, Genetics and Evolution 11 (2011) 789–797 793 Fig. 3. Evolution Canyons diagram simplified from Sikorski and Nevo (2007); the origin of B. simplex samples is coded by geometric shape (circles for Canyon 1, squares for Canyon 2) and color (blue-green for north-facing slopes, orange for south-facing slopes); generalized phenotypic similarity between the various groups of samples is represented in the insert box, top right, showing that similarity correlates with type of slope of origin rather than canyon of origin. basis of purely phenotypic criteria. B. anthracis was described as mentioned earlier in relation to the pathogenesis of anthrax disease. B. cereus was described as a microbe commonly associated with cereals (Frankland and Frankland, 1887). During the same period, B. mycoides was described on the basis of the striking rhizoid morphology its colonies exhibit during growth on nutrient media (Flügge, 1886). Thirty years later, B. thuringiensis was described based on its production of parasporal protein crystals and their associated entomocidal properties (Berliner, 1915). However, the emergence of DNA hybridization techniques, then 16S rDNA and multilocus sequence typing (MLST; http://mlstoslo.uio.no), progressively revealed that these species were much more closely related than what their taxonomical status implied. 16S rDNA provided insufficient resolution at the species level and while several MLST schemes proved useful for distinguishing clades, they could not resolve the traditional species either. The concept of a B. cereus sensu lato group drawing these species tightly together was therefore introduced about a decade ago to acknowledge that proximity, with the qualifier sensu stricto added to ‘‘regular’’ B. cereus strains to distinguish them from the group name (Léonard et al., 1997). At roughly the same time, two additional species were described as members of the group: B. pseudomycoides as a genetic variant of B. mycoides (Nakamura, 1998) and B. weihenstephanensis as a psychrotrophic variant of B. cereus s. s. (Lechner et al., 1998). Most recently a cluster of thermophilic strains of clinical origin was proposed to represent a new species, to be named Bacillus cytotoxicus (Lapidus et al., 2008). These strains are still referred to as B. cereus subsp. cytotoxis in the literature and public databases pending official approval of the new species designation. There is in fact a growing consensus in the research community that the members of the B. cereus group should be considered as forming one single species from which different ecotypes and pathotypes emerge in a dynamic fashion, leading in some cases to the formation of clonal complexes with specific phenotypes (Helgason et al., 2000; Priest et al., 2004; Rasko et al., 2005; Tourasse et al., 2006). This view stems from crucial insights obtained over the last decade from the combination of MLST, genomics and the study of large plasmids. Indeed, we now know that the main phenotypical properties that were originally used to distinguish B. cereus s. s., B. thuringiensis and B. anthracis are directly related to the presence or absence of large plasmids, which carry the genetic determinants that are responsible for those phenotypes. In B. anthracis, the virulence plasmids pXO1 (192 kb) and pXO2 (96 kb) encode the anthrax toxin and capsule genes, respectively, as well as associated regulatory elements (Koehler, 2009). In B. thuringiensis there can be one or several so-called ‘Cry plasmids’, most of them conjugative, that encode insecticidal crystal toxins (Schnepf et al., 1998); B. thuringiensis strains that have lost their toxin-bearing plasmids are effectively indistinguishable from B. cereus s. s. strains. B. cereus s. s. strains encode a range of toxins and other extracellular virulence factors on the chromosome, but it has been shown that the same genes are present and actively expressed in B. thuringiensis strains (Swiecicka et al., 2006). Fig. 4 shows a conceptualized view of the B. cereus sensu lato phylogenomic structure based on MLST data from multiple studies integrated in the HyperCAT database (Tourasse et al., 2006, 2010). Various methods of molecular typing have periodically been put forward with the claim that they could differentiate B. cereus s. s. and B. thuringiensis, but the results from genomic studies have essentially put to rest the idea of them forming two genetically distinct species. B. cereus s. s. and B. thuringiensis had long been known to be highly polymorphic, and what the genomics showed was that the amount of variation within and overlap between each of these so-called species is such that neither one displays a coherent individual phenotypic or genomic identity in comparison to the other beyond plasmid-borne virulence properties (Rasko et al., 2005). The ‘‘B. cereus vs. B. thuringiensis’’ problem (i.e., that B. thuringiensis exists as a separate species on paper only) raises questions of policy that extend beyond the realm of scientific accuracy. Until now, the separate species status of B. thuringiensis has been a key point in the safety evaluation of strains used in commercial biopesticide formulations, which represent a worldwide for-profit industry. In the USA, the regulatory process for the registration of biopesticidal strains is currently being updated by the Environmental Protection Agency to address concerns regard- 794 H. Maughan, G. Van der Auwera / Infection, Genetics and Evolution 11 (2011) 789–797 Fig. 4. Phylogenomic structure of the B. cereus group stylized as an unrooted tree diagram based on HyperCAT (Tourasse et al., 2010); top-level clusters identified in HyperCAT are represented as leaves and identified by roman numerals (I–VII); classical species designations indicate the dominant contents of each cluster according to the established pre-genomic taxonomy; topology of relationships between clusters is respected but branch length and leaf size are not necessarily to scale, and branching within leaves is shown for aesthetic purposes only. Note that the B. anthracis complex is shown as a budding sub-group colored in red to emphasize its close relationship to B. cereus group strains in Cluster III. Source data may be accessed at: http://mlstoslo.uio.no. ing potential toxin content, but to date the ‘‘old’’ taxonomy is still used as a basis for strain classification. What this means for the reliability of the process and safety of registered products is still unclear, but certainly warrants further investigation. At first glance B. anthracis appears to pose less of a taxonomical problem, because in its canonical form it is highly monomorphic and possesses clearly identifiable phenotypic and genetic chromosomal features (Keim et al., 1999; Kolstø et al., 2009; Van Ert et al., 2007). Specifically, the key chromosomal feature is a nonsense mutation in the gene encoding PlcR (Easterday et al., 2005; Gohar et al., 2008), a master regulator that is responsible for numerous phenotypic traits in all B. cereus s. l. group organisms. In B. anthracis, the plcR gene is inactivated by a single point mutation and the master regulator is AtxA, which is encoded by the atxA gene on pXO1 (Fouet and Mock, 2006; Mignot et al., 2001). Combined with prophage content (Sozhamannan et al., 2006), sensitivity to gamma phage (Abshire et al., 2005; Schuch et al., 2002) and the presence of the pXO1 and pXO2 virulence plasmids, this was for a time thought to be sufficient to characterize B. anthracis unambiguously (Klee et al., 2006). However, the phage-based criteria have since been shown to be non-exclusive (Marston et al., 2006), and recent studies have shown that plasmids closely related to pXO1 and pXO2 can be found widely in environmental isolates of B. cereus sensu lato (Bahl and Rosenberg, 2010; Hu et al., 2009). In addition, a number of strains of B. cereus s. s. and B. thuringiensis, many of them pathology-associated clinical isolates, have been found displaying phenotypic characteristics of B. anthracis (including production of anthrax toxins) and, significantly, containing plasmids very closely related to the anthrax virulence plasmids pXO1 and pXO2 (Hoffmaster et al., 2004; Klee et al., 2010, 2006; Okinaka et al., 2006; Pannucci et al., 2002a,b). Genomic analyses revealed that these strains cluster closer to the canonical B. anthracis clonal complex than to the bulk of B. cereus s. s./B. thuringiensis strains, although they do not possess the point mutation that inactivates PlcR. However, not all B. cereus strains that are genomically very close to B. anthracis necessarily contain pXO1-like and/or pXO2like plasmids. The existence of all these so-called ‘‘close neighbors’’ of B. anthracis demonstrates a degree of phylogenomic continuity that can be argued to invalidate the maintenance of B. anthracis as a separate species in purely genetic terms. How to deal with these issues is the subject of much debate. One proposal under consideration is for strains possessing the canonical PlcR mutation and select virulence factors to be classified as B. anthracis sensu stricto, while borderline isolates lacking those characteristics would be classified as B. anthracis sensu lato (Okinaka et al., 2006). Another is for those borderline isolates to be classified as B. cereus var. anthracis (Kolstø et al., 2009), which H. Maughan, G. Van der Auwera / Infection, Genetics and Evolution 11 (2011) 789–797 could be subjected to specific regulatory controls without leading to an overhaul of safety grading for the rest of the B. cereus s. l. group. Current trends in the published record as well as discussion in the B. cereus research community suggest it is unlikely that the classification of B. cereus s. l. group organisms will be changed to single-species status in the foreseeable future. It seems that, for now, we will keep the notion of a B. cereus s. l. group as a ‘‘wrapper’’ and attempt to work around the problems within by defining genomic criteria that allow rational management of ‘‘science vs. policy’’ issues. 795 identification of closely related strains/species, which could then be followed by the more in-depth genomic characterization of interesting groups of strains. Such a study has recently been done in B. subtilis using microarray and sequencing technologies and has uncovered a great deal of genomic diversity within this group of closely related B. subtilis strains (Earl et al., 2007, 2008). Although this may currently seem unreasonable to do with all bacterial species of interest, advances in sequencing technologies coupled with decreasing costs, in addition to advances in single-cell genomics technologies and metagenomics methods, should eventually enable a thorough description and classification of the amazing molecular and ecological diversity of Bacillus. 5. Reconciling molecules and ecology In the previous examples it is important to note that 16S-rDNA based taxonomy does not disagree with methods offering more resolution, but is simply unable to accurately estimate the ecological diversity present within identical sequences. Let us now conduct a brief ‘‘reductio ad absurdum’’ experiment to estimate the number of Bacillus species based on ecology. As discussed above, it is essentially meaningless to treat B. cereus and B. thuringiensis as distinct species, at least in genetic terms. However, for this exercise we will purposefully ignore that fact, and proceed to calculate the average pairwise nucleotide difference between 16S rDNA sequences identified in the RDP database as belonging to B. cereus (n = 513), B. thuringiensis (n = 277), or B. anthracis (n = 136). We then use the resulting value of 0.7% difference as a cutoff to predict the number of ecologically distinct Bacillus species that could be said to be currently represented in the sequence databases if the B. cereus group species distinctions were considered valid and an appropriate yardstick for the rest of the genus. The first striking observation we make is that the divergence between these three species is statistically insignificant. For example, many B. cereus isolates are more closely related to B. anthracis isolates than they are to other B. cereus strains, basically confirming that these are not different species. But if we ignore this issue for now and apply the 0.7% divergence to all 7510 Bacillus 16S sequences we predict the existence of 1034 species of Bacillus, an almost tenfold increase from our earlier prediction of 116 using the conventional cutoff of 97% nucleotide identity. There are two ways of interpreting this order of magnitude increase in number of species predicted. On the one hand, this may mean we are vastly underestimating the number of ecologically distinct species of Bacillus species in nature by using cutoffs that are too conservative. On the other, this brings us back to the fundamental flaw of defining species on the basis of ecological traits that may not be congruent with phylogenomic structure. In fact, these interpretations are not mutually exclusive, and it is likely that the Bacillus classification, in its current state, is misleading in both directions. Because the ecology of most species is virtually unknown and diversity in those areas of the tree is estimated exclusively on the basis of 16S rDNA sequence, we are probably missing out on a lot of ecological richness. At the same time, we are blinded to the extreme genomic proximity of historically defined species such as those in the B. cereus group where phenotypes were mistakenly assumed to follow phyletic divergences. What is the solution? Clearly molecular based taxonomy is the way forward, but significant improvements can be made by the addition of ecological data as in the case of B. simplex and the B. cereus group. It is unlikely that truly relevant ecological data will be obtained by present culturing methods, leaving us to predict ecology based on site of isolation and further genomic and metagenomic sequencing efforts. For example, sequencing of 16S rDNA or other conserved loci can be used for initial clustering and Acknowledgements We wish to thank Dr. Ashlee M. 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